Water soluble nitric oxide-releasing polyglucosamines and uses thereof

ABSTRACT

The presently disclosed subject matter provides nitric oxide-releasing polysaccharides and oligosaccharides, in particular, polyglucosamines, and their use in biomedical and pharmaceutical applications. More particularly, in some embodiments, the presently disclosed subject matter provides nitric oxide-releasing polysaccharides and oligosaccharides that release nitric oxide in a controlled and targeted manner, thereby prolonging the therapeutic effects of nitric oxide and improving the specificity of nitric oxide delivery to targeted cells and/or tissues.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/421,525, filed Feb. 13, 2015, which is a 35 U.S.C. § 371 nationalphase entry of International Application No. PCT/US2013/055360, filedAug. 16, 2013, and published in English on Feb. 20, 2014, asInternational Publication No. WO 2014/028847, and which claims thebenefit of U.S. Provisional Application No. 61/684,373, filed Aug. 17,2012, the disclosures of each of which are incorporated by referenceherein in their entirety.

GOVERNMENT INTEREST

This invention was made with government support under EB000708 awardedby The National Institutes of Health. The Government has certain rightsin the invention.

FIELD OF THE INVENTION

The presently disclosed subject matter provides nitric oxide-releasingpolysaccharides and oligosaccharides, in particular, polyglucosamines,and their use in biomedical and pharmaceutical applications. Moreparticularly, in some embodiments, the presently disclosed subjectmatter provides nitric oxide-releasing oligosaccharides that releasenitric oxide in a controlled and targeted manner, thereby prolonging thetherapeutic effects of nitric oxide and improving the specificity ofnitric oxide delivery to targeted cells and/or tissues.

BACKGROUND OF THE INVENTION

The discovery of the multifaceted role of nitric oxide (NO) in biology,physiology, and pathophysiology, see Marietta, M. A., at al.,BioFactors, 2, 219-225 (1990), has led to the search for nitric oxidedonors capable of controlled nitric oxide release. See Keefer, L. K.,Chemtech, 28, 30-35 (1998). To date, researchers have discovered that NOregulates a range of biological processes in the cardiovascular,gastrointestinal, genitourinary, respiratory, and central and peripheralnervous systems. See Ignarro, L. J., Nitric Oxide: Biology andPathobiology; Academic Press: San Diego, 2000; and Ignarro, L. J. etal., Proc. Natl. Acad. Sci., USA., 84, 9265-9269 (1987). Furthermore,the discovery of NO as a vasodilator and its identification as both anantibiotic and a tumoricidal factor have made NO an attractivepharmaceutical candidate. See, for example, Radomski, M. W., et al., Br.J. Pharmacol., 92, 639-646 (1987); Albina, J. E., and Reichner, J. S.;Canc. Metas. Rev., 17, 19-53 (1998); Nablo, B. J., at al., J. Am. Chem.Soc., 123, 9712-9713 (2001); Cobbs, C. S., et al., Cancer Res., 55,727-730 (1995); Jenkins, D. C., at al., Proc. Natl. Acad. Sci., USA.,92, 4392-4396 (1995); and Thomsen, L. L., et al., Br. J. Cancer., 72,41-44 (1995). Several nitric oxide donors have been reported, the mostnotable being N-diazeniumdiolates. Generally, N-diazeniumdiolate NOdonors are small molecules synthesized by the reaction of amines with NOat elevated pressure and have been used, for example, to spontaneouslygenerate NO in aqueous solution. See Hrabie, J. A. and Keefer, L. K.,Chem, Rev., 102, 1135-1154 (2002).

Therapeutic strategies to explore the activities of nitric oxide donors,for example, to kill tumor cells, are problematic in part because thenitric oxide delivery systems known in the art release or donate nitricoxide indiscriminately. Thus, there is a need in the art for a nitricoxide delivery system that releases or donates nitric oxide in acontrolled and/or targeted manner to facilitate an improvedunderstanding of the function of NO in physiology and to provide for thedevelopment of NO-associated therapies.

SUMMARY OF THE INVENTION

In embodiments, the subject matter disclosed herein is directed to apolyglucosamine that contains a covalently bound nitric oxide releasingmoiety, i.e. a NO donor. At least one structural unit in thepolyglucosamine backbone contains the structural unit of formula I.Optionally, at least one structural unit of the polyglucosamine furthercomprises the structural unit of formula II. Advantageously, thepolyglucosamine described herein is water soluble and is capable ofdelivering NO to a target.

In embodiments, the subject matter disclosed herein is directed tomethods of delivering or releasing NO to a subject comprisingadministering a polyglucosamine comprising at least one structural unitof formula I and optionally, further comprising at least one structuralunit of formula II.

In embodiments, the subject matter disclosed herein is directed tomethods of treating a disease state in a subject comprisingadministering a polyglucosamine comprising at least one structural unitof formula I and optionally, further comprising at least one structuralunit of formula II.

In embodiments, the subject matter disclosed herein is directed to apharmaceutical composition comprising a polyglucosamine containing atleast one structural unit of formula I and optionally, furthercomprising at least one structural unit of formula II and apharmaceutically acceptable carrier, excipient or diluent.

In embodiments, the subject matter disclosed herein is directed tomethods of preparing a polyglucosamine comprising at least onestructural unit of formula I and optionally, further comprising at leastone structural unit of formula II.

In another embodiment, the subject matter disclosed herein is directedto a method of disrupting, eradicating or preventing a biofilm byemploying a polyglucosamine comprising at least one structural unit offormula I and optionally, further comprising at least one structuralunit of formula II.

The subject matter is described fully in the drawings herein and in thespecification set forth below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 describes an advantageous property of the presently disclosedfunctionalized polyglucosamines.

FIG. 2 shows the percent of C, H and N found in certain embodiments.

FIG. 3 is a ¹H NMR of certain embodiments.

FIG. 4A depicts the release profile of certain chitosan oligosaccharidesCSO 2-NO in different NO conjugation solvents. Methanol:H₂O 7:3 resultedin total NO storage of 0.87 μmol/mg; FIG. 4B depicts specific datapoints for the nitric oxide release profiles of Chitosan 2/NO-5k inmethanol (solid square), methanol/water 9:1 (solid circle), 8:2 (opentriangle), 7:3 (solid triangle), and 6:4 v/v (open square).

FIGS. 5A & 5B depicts (A) Real time NO release profiles for certainNO-releasing chitosan oligosaccharides; and (B) plot of t[NO] vs timefor certain NO-releasing chitosan oligosaccharides.

FIG. 6 shows the t[NO], [NO]_(max), and t_(1/2) of certain embodiments.

FIG. 7 depicts the data showing NO-releasing CSO led to 5-log reductionof P. aeruginosa bacteria with biofilms. Anti-biofilm efficacy ofNO-releasing (solid symbols) and control (open symbols) chitosanoligosaccharides (Chitosan 1-5k; CSO 1-NO (sphere), Chitosan 2-5k; CSO2-NO (square), and Chitosan 3-5k; CSO 3-NO (triangle)) againstestablished P. aeruginosa biofilms. Control chitosan oligosaccharidesresulted in no significant reduction in bacteria viability.

FIGS. 8A and B depict cytotoxicity in L929 mouse fibroblast. Theviability of L929 mouse fibroblasts exposed to control and NO-releasingchitosan oligosaccharides at concentration for 5-log bacteria viabilityreduction (MBC) against P. aeruginosa biofilms. Each parameter wasanalyzed with multiple replicates (n=3).

FIG. 9A-F depict confocal fluorescence images of RITC-labeled chitosanoligosaccharide association with P. aeruginosa in biofilms (A. Chitosan2/NO-5k, B. Chitosan 3/NO-5k, C. Chitosan 2-10k) and images of syto 9labeled biofilms incubated with D) Chitosan 2/NO-5k, E) Chitosan 3/NO-5kand F) Chitosan 2/NO-10k. Green fluorescence of syto 9 indicates the P.aeruginosa bacteria embedded in the biofilms. Red fluorescence of RITCindicates the association of RITC-labeled chitosan oligosaccharides withP. aeruginosa in biofilms. Scale bar: 40 μm.

FIG. 10A-H depict Bright field and fluorescent images of RITC-modifiedChitosan 2/NO-5k at A) 24, B) 28, C) 42 min and Chitosan 3/NO-5k at D)82, E) 86, F) 110, H) 120 min (150 μg mL⁻¹) association with P.aeruginosa. Overlay images of P. aeruginosa incubated with G) Chitosan2/NO-5k at 44 min and H) Chitosan 2/NO-5k at 120 min.

DETAILED DESCRIPTION

The presently disclosed nitric oxide (NO) releasing polyglucosamines,also referred to in embodiments as NO-releasing chitosanoligosaccharides, are advantageously water soluble and tunable. Theseproperties contribute to the usefulness of the presently disclosedpolyglucosamines in therapeutics and disease states where water solubletherapeutics are advantageous, for example, in the treatment of cysticfibrosis. Other advantages over known NO releasing particles that thepresently disclosed functionalized NO releasing polyglucosamines possessinclude: 1. Distinct synthesis routes and chemical composition bygrafting secondary amine-containing oligomer chains onto chitosanoligosaccharides; 2. Tunability of NO storage and NO-release kinetics isan advantage. By tuning the number of secondary amines on the aziridineoligomer side chains, NO storage can be controlled. Furtherfunctionalization of the amines on the resulting materials by compoundsof different hydrophobicity/hydrophilicity enables the control overNO-release kinetics. Indeed, much larger NO storage was yielded by thepresently disclosed functionalized polyglucosamines; and 3. In contrastto particles, the functionalized polyglucosamines described herein arewater soluble, facilitating a wider range of applications includingbiomedical application where water-solubility is desired. A previouslydisclosed NO-releasing chitosan (U.S. Pat. No. 6,451,337) is not watersoluble. This highlights another advantage that the presently disclosedwater soluble functionalized can readily penetrate and eradicatebiofilms.

The present invention can be embodied in different forms and should notbe construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. For example, features illustrated with respect toone embodiment can be incorporated into other embodiments, and featuresillustrated with respect to a particular embodiment can be deleted fromthat embodiment. In addition, numerous variations and additions to theembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference herein in their entirety.

As used herein, “a,” “an,” or “the” can mean one or more than one. Forexample, “a” NO releasing moiety can mean a single or a multiplicity.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

The term “consists essentially of” (and grammatical variants), asapplied to the compositions of this invention, means the composition cancontain additional components as long as the additional components donot materially alter the composition.

The term “treatment effective amount” or “effective amount,” as usedherein, refers to that amount of a functionalized polyglucosamine thatimparts a modulating effect, which, for example, can be a beneficialeffect, to a subject afflicted with a disorder, disease or illness,including improvement in the condition of the subject (e.g., in one ormore symptoms), delay or reduction in the progression of the condition,prevention or delay of the onset of the disorder, and/or change inclinical parameters, disease or illness, etc., as would be well known inthe art. For example, a therapeutically effective amount or effectiveamount can refer to the amount of a composition, compound, or agent thatimproves a condition in a subject by at least 5%, e.g., at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 100%.

“Treat” or “treating” or “treatment” refers to any type of action thatimparts a modulating effect, which, for example, can be a beneficialeffect, to a subject afflicted with a disorder, disease or illness,including improvement in the condition of the subject (e.g., in one ormore symptoms), delay or reduction in the progression of the condition,and/or change in clinical parameters, disease or illness, etc., as wouldbe well known in the art.

The terms “disrupting” and “eradicating” refer to the ability of thepresently disclosed functionalized polyglucosamines to combat biofilms.The biofilms may be partially eradicated or disrupted, meaning that thecells no longer attach to one another or to a surface. The biofilm maybe completely eradicated, meaning that the biofilm is no longer aninterconnected, cohesive or continuous network of cells to a substantialdegree.

As used herein, the term “water soluble” means that the functionalizedpolyglucosamine is more soluble in water at room temperature than thepolyglucosamine before functionalization. Preferably, water solublefunctionalized polyglucosamines disclosed herein are soluble suchthat >50 mg of functionalized polyglucosamine dissolves per mL of water.More preferably, water soluble functionalized polyglucosamines disclosedherein are soluble such that >75 mg of functionalized polyglucosaminedissolves per mL of water. Most preferably, water soluble functionalizedpolyglucosamines disclosed herein are soluble such that >100 mg offunctionalized polyglucosamine dissolves per mL of water.

The terms “nitric oxide donor” or “NO donor” refer to species thatdonate, release and/or directly or indirectly transfer a nitric oxidespecies, and/or stimulate the endogenous production of nitric oxide invivo and/or elevate endogenous levels of nitric oxide in vivo such thatthe biological activity of the nitric oxide species is expressed at theintended site of action.

The terms “nitric oxide releasing” or “nitric oxide donating” refer tomethods of donating, releasing and/or directly or indirectlytransferring any of the three redox forms of nitrogen monoxide (NO+,NO⁻, NO). In some cases, the nitric oxide releasing or donating isaccomplished such that the biological activity of the nitrogen monoxidespecies is expressed at the intended site of action.

The term “microbial infection” as used herein refers to bacterial,fungal, viral, and yeast infections.

The “patient” or “subject” treated in the many embodiments disclosedherein is desirably a human patient, although it is to be understoodthat the principles of the presently disclosed subject matter indicatethat the presently disclosed subject matter is effective with respect toall vertebrate species, including mammals, which are intended to beincluded in the terms “subject” and “patient.” Suitable subjects aregenerally mammalian subjects. The present invention finds use inresearch as well as veterinary and medical applications. The term“mammal” as used herein includes, but is not limited to, humans,non-human primates, cattle, sheep, goats, pigs, horses, cats, dog,rabbits, rodents (e.g., rats or mice), etc. Human subjects includeneonates, infants, juveniles, adults and geriatric subjects.

The subject can be a subject “in need of” the methods disclosed hereincan be a subject that is experiencing a disease state and/or isanticipated to experience a disease state, and the methods andcompositions of the invention are used for therapeutic and/orprophylactic treatment.

The oligosaccharide described herein are polyglucosamines.Polyglucosamines and derivatives thereof are known in the as chitosansand derivatives thereof. Particularly useful polyglucosamines arepolymers that can range in size 100 to 20,000 g/mol. Smallerpolyglucosamines having molecular weights below 100 g/mol and largerones having molecular weights above 20,000 g/mol are also contemplated.Chitosans having a molecular weight above 20,000 g/mol may need to befurther functionalized to be water soluble. An important feature ofuseful polyglucosamines is an available nitrogen on the carbohydratebackbone that is derivatized according to the methods described hereinto form a NO-releasing polyglucosamine. Advantageously, thepolyglucosamines disclosed herein are water soluble.

Chitosan is a linear polysaccharide composed of randomly distributedβ-(1-4)-linked D-glucosamine (deacetylated unit) andN-acetyl-D-glucosamine (acetylated unit). It is a polyglucosamine.Chitosan is derived from chitin, a polysaccharide found in theexoskeleton of shellfish such as shrimp, lobster, and or crabs. It hasthe following structure:

Chitosan is biodegradable and biocompatible. Chitosan itself is onlysoluble under acidic conditions. Chitosan polysaccharides are insolubleunder physiological conditions. Additionally, their NO storage is rathermodest (˜0.2 μmol/mg) likely due to poor solubility of polysaccharidesin basic solutions necessary for NO donor formation. Valmikinathan, C.M.; Mukhatyar, V. J.; Jain, A.; Karumbaiah, L.; Dasari, M.; Bellamkonda,R. V. Photocrosslinkable chitosan based hydrogels for neural tissueengineering. Soft Matter 2012, 8, 1964-1976; Zhang, J. L.; Xia, W. S.;Liu, P.; Cheng, Q. Y.; Tahirou, T.; Gu, W. X.; Li, B. ChitosanModification and Pharmaceutical/Biomedical Applications. Mar. Drugs2010, 8, 1962-1987; Wan, A.; Gao, Q.; Li, H. L. Effects of molecularweight and degree of acetylation on the release of nitric oxide fromchitosan-nitric oxide adducts. J. Appl. Polym. Sci. 2010, 117,2183-2188. To synthesize N-diazeniumdiolate-functionalized chitosanderivatives with improved NO storage, we prepared water-soluble chitosanoligosaccharides by the degradation of chitosan polysaccharides inhydrogen peroxide. A benefit of the chitosan oligosaccharides describedherein are relatively low-molecular weight from 100 to 20,000 g/mol, inparticular about 10,000 g/mol or less; or about 8000 g/mol or less; orabout 5000 g/mol or less; or about 2,500 g/mol or less and their abilityto readily penetrate biofilms. Maghami, G. G.; Roberts, G. A. F.Evaluation of the viscometric constants for chitosan. Makromol. Chem.1988, 189, 195-200; Porporatto, C.; Bianco, I. D.; Riera, C. M.; Correa,S. G., Chitosan induces different L-arginine metabolic pathways inresting and inflammatory macrophages. Biochem. Biophy. Res. Comm. 2003,304, 266-272. Chitosan oligosaccharides described herein have greater NOstorage of up to about 8.7 μmol/mg and are also soluble under neutraland basic conditions.

The primary amino groups on the backbone of chitosan are chemicalhandles for the preparation of the NO-releasing oligosaccharidesdisclosed herein. As shown in the schemes below, secondary amino groupsare prepared from the primary amino groups.

Useful agents to form the secondary amino groups are selected from thegroup consisting of aziridines, in particular 2-methyl aziridine, andthiiranes and the like.

In an embodiment, the subject matter disclosed herein is directed to apolyglucosamine, e.g., a chitosan oligosaccharide comprising at leastone structural unit of formula I:

and optionally, at least one structural unit of formula II:

wherein,

-   -   R₁, R₂, R₃ and R₄, if present, are each independently selected        from the group consisting of hydrogen; C₁₋₅ alkyl(C═O)—, when        the C₁₋₅ alkyl is methyl, Me(C═O)— is an acyl, Ac; and C₁₋₅        alkyl;    -   , in each instance, is a single or double bond,    -   wherein in each instance where it is a double bond, R₁, R₂, R₃        or R₄ attached to the double bond-O is absent; when R₁ is        absent, R₅ is hydrogen, hydroxyl, C₁₋₅ alkyl or C₁₋₅ alkoxy;        when R₃ is absent, R₆ is hydrogen, hydroxyl, C₁₋₅ alkyl or C₁₋₅        alkoxy;    -   wherein in each instance where it is a single bond, R₁, R₂, R₃        or R₄ attached to the double bond-O is present; when R₁ is        present, R₅ is hydrogen;

when R₃ is present, R₆ is hydrogen;

-   -   Q is —(CR_(c)R_(d))_(v)—;        -   wherein R_(c) and R_(d) are independently hydrogen or C₁₋₅            alkyl, such as methyl, ethyl, n-propyl, isopropyl, t-butyl,            n-butyl, isobutyl and pentyl. Preferably, R_(c) and R_(d)            are independently hydrogen, methyl or ethyl; and v is an            integer from 2 to 6; preferably, v is 2;    -   p is an integer from 1 to 100, preferably 1 to 50; more        preferably 1 to 25; most preferably 1 to 10;    -   A is

-   -   -   wherein, L is S, O or N; and        -   G, in each instance, is independently, hydrogen, or is taken            together with L to form a nitric oxide donor;

    -   X is hydrogen, C₁₋₅ alkyl or is taken together with N to form a        nitric oxide donor;

    -   B is hydrogen or —Y—Z, wherein Y is a spacer and Z is a polymer        or a terminus group; or B is absent;

    -   D is —NR_(a)R_(b), wherein R_(a) and R_(b) are independently        selected from the group consisting of hydrogen, formyl, C₁₋₅        alkyl(C═O)—, when the C₁₋₅ alkyl is methyl, Me(C═O)— is an acyl,        Ac, C₁₋₅ alkyl and C₁₋₅ alkyl ester;

    -   or D is

Useful values of R₁, R₂, R₃ and R₄, if present, are each independentlyselected from the group consisting of hydrogen and C₁₋₅ alkyl. When oneor more of R₁, R₂, R₃ and R₄ is C₁₋₅ alkyl, it is selected from methyl,ethyl, n-propyl, isopropyl, t-butyl, n-butyl, isobutyl and pentyl.Preferably, R₁, R₂, R₃ and R₄, if present, is hydrogen or methyl. Mostpreferably, R₁, R₂, R₃ and R₄, if present, is hydrogen.

In all embodiments,

, in each instance, is a single or double bond. Preferably, it is asingle bond in all instances.

Q is —(CR_(c)R_(d))_(v)—; wherein R_(c) and R_(d) are independentlyhydrogen or C₁₋₅ alkyl, such as methyl, ethyl, n-propyl, isopropyl,t-butyl, n-butyl, isobutyl and pentyl. Preferably, R_(c) and R_(d) areindependently hydrogen, methyl or ethyl. Useful values of v are integersfrom 2 to 6. Preferably, v is 2.

Useful values of p include any integer from 1 to 100. Preferably p is aninteger from 1 to 50. More preferably, p is an integer from 1 to 25.Most preferably, p is an integer from 1 to 10, such as, 1, 2, 3, 4, 5,6, 7, 8, 9 and 10.

Useful values of L are N, S and O. Preferably, L is N or S. In eachinstance that G occurs, it is independently hydrogen or a nitric oxidedonor. Since the nitric oxide donor contributes to the amount ofavailable NO on the polyglucosamine, it is preferable that G is a nitricoxide donor. In preferred embodiments, at least 30% of G present on apolyglucosamine is a nitric oxide donor. More preferably, at least 50%of G present on a polyglucosamine is a nitric oxide donor. Even morepreferably, at least 90% of G present on a polyglucosamine is a nitricoxide donor. Most preferably, at least 95% of G present on apolyglucosamine is a nitric oxide donor.

Useful values of X are hydrogen, C₁₋₅ alkyl or a nitric oxide donor.Since the nitric oxide donor contributes to the amount of available NOon the polyglucosamine, it is preferable that X is a nitric oxide donor.In preferred embodiments, at least 30% of X present on a polyglucosamineis a nitric oxide donor. More preferably, at least 50% of X present on apolyglucosamine is a nitric oxide donor. Even more preferably, at least90% of X present on a polyglucosamine is a nitric oxide donor. Mostpreferably, at least 95% of X present on a polyglucosamine is a nitricoxide donor.

Useful values of B are hydrogen, —Y—Z, wherein Y is a spacer and Z is amonomer or polymer, or B is a terminus group. B may also be absent whenL is O or S. As used herein, a terminus group is any end-capping groupat the terminus of a polymer or monomer. These groups are known in theart. Preferably, when B is a terminus group, it is hydrogen, hydroxyl orC₁₋₅ alkyl.

Useful values of Z include monomers and polymers known in the art,especially those used in active pharmaceutical ingredients. Particularlyuseful polymers or monomers include:

wherein j, in each instance, is an integer from 1 to 100.

Useful spacers, Y, in the formulae disclosed herein include spacers orlinkers known in the art, especially those used in active pharmaceuticalingredients. Particularly useful spacers include the following:

wherein, R_(p), R_(q), and R_(t), in each instance, are independently,hydrogen or hydroxyl; and k is an integer from 1 to 20.

Using the strategies disclosed herein, any secondary amino group presenton the oligosaccharide can be modified as described herein to form aNO-releasing oligosaccharide. The secondary amino groups attacheddirectly to the sugar backbone moieties or secondary amino groupspendant on the backbone sugar moieties can be functionalized with a NOreleasing moiety. As disclosed fully herein in the synthetic routes,primary amines are modified to secondary amines. This modification canbe facilitated by aziridines, thiiranes and the like.

Useful NO releasing moieties include any NO releasing group known in theart. Particularly useful are residues of NO releasing groups, i.e. NOdonors, are covalently bound to N, S or O on the polyglucosamine. The NOdonor is taken together with the atom on the polyglucosamine to which itis bound to form a moiety selected from the group consisting of adiazeniumdiolate, —NO as part of a nitrosothiol group for example, anitrosamine, a hydroxyl nitrosamine, a hydroxyl amine, a hydroxyurea,and combination thereof. Preferably, the NO releasing moiety is adiazeniumdiolate. These groups may be present in the form of a salt.

In some embodiments, the NO donor is a N-diazeniumdiolate (i.e., a1-amino-substituted deazen-1-lum-1,2-diolate), N-Diazeniumdiolates areparticularly attractive as NO donors due to their ability to generate NOspontaneously under biological conditions. See Hrabie, J. A. and Keefer,L. K., Chem. Rev., 102, 1135-1154 (2002); and Napoli, C. and Ianarro, L.J., Annu. Rev. Pharmacol. Toxicol., 43, 97-123 (2003). SeveralN-diazeniumdiolate compounds have been synthesized using a range ofnucleophilic residues that encompass primary and secondary amines,polyamines, and secondary amino acids, See Hrabie, J. A., and Keefer L.K., Chem. Rev., 102, 1135-1154 (2002). In the formation of theN-diazeniumdiolate, one equivalent of amine reacts with two equivalentsof nitric oxide under elevated pressure. A base (e.g., an alkoxide likemethoxide) removes a proton from the amine nitrogen to create theanionic, stabilized [N(O)NO] group. While stable under ambientconditions, N-diazeniumdiolates decompose spontaneously in aqueous mediato generate NO at rates dependent upon pH, temperature, and/or thestructure of the amine moiety. For example, N-diazeniumdiolate-modifiedproline (PROLI/NO), 2-(dimethylamino)-ethylputreamine (DMAEP/NO),N,N-dimethylhexanediamine (DMHD/NO), and diethylenetriamine (DETA/NO)have been developed as small molecule NO donors with diverse NO releasehalf-lives ranging from 2 seconds to 20 hours at pH 7.4 and 37° C. SeeHrabie, J. A., and Keefer, L. K., Chem. Rev., 102, 1135-1154 (2002); andKeefer, L. K., Annu, Rev. Pharmacol. Toxicol 43, 585-607 (2003).

The secondary amine functional group of the polyglucosamine is convertedin high yields to a nitric oxide donor in the presence of a strong baseand gaseous nitric oxide. As provided herein, the solvent system canaffect the charging of the polyglucosamine with NO.

In an embodiment, when the polyglucosamine is specificallyfunctionalized with an aziridine as described herein, the functionalizedpolyglucosamine comprises at least one structural unit of formula Ia:

and optionally, at least one structural unit of formula IIa:

wherein, the definitions of the variables are as defined above with theexception of D. In this embodiment, D is —NR_(a)R_(b), wherein R_(a) andR_(b) are independently selected from the group consisting of hydrogen,formyl, C₁₋₅ alkyl(C═O)—, C₁₋₅ alkyl and C₁₋₅ alkyl ester; or D has thestructure:

wherein, X, Q, p and B are as described above.

In an aspect of this embodiment, when the polyglucosamine isspecifically functionalized with an aziridine as described herein, thefunctionalized polyglucosamine comprises at least one structural unit offormula VIII:

wherein, the variables are as described above for this embodiment.

The term “amino” and “amine” refer to nitrogen-containing groups such asNR₃, NH₃, NHR₂, and NH₂R, wherein R can be as described elsewhereherein. Thus, “amino” as used herein can refer to a primary amine, asecondary amine, or a tertiary amine. In some embodiments, one R of anamino group can be a diazeniumdiolate (i.e., NONO).

The terms “cationic amine” and “quaternary amine” refer to an aminogroup having an additional (i.e., a fourth) group, for example ahydrogen or an alkyl group bonded to the nitrogen. Thus, cationic andquaternary amines carry a positive charge.

The term “alkyl” denotes a straight or branched hydrocarbon chaincontaining 1-24 carbon atoms, e.g., 1-12 carbon atoms. Examples of alkylgroup include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl, and the like.

The term “alkoxy” is used herein to mean a straight or branched chainalkyl radical, as defined above, unless the chain length is limitedthereto, bonded to an oxygen atom, including, but not limited to,methoxy, ethoxy, n-propoxy, isopropoxy, and the like. Preferably thealkoxy chain is 1 to 5 carbon atoms in length, more preferably 1-3carbon atoms in length.

The following specific embodiments are disclosed:

-   -   1. A polyglucosamine (chitosan oligosaccharide) comprising,    -   at least one structural unit:

-   -   and optionally, at least one structural unit:

-   -   wherein,        -   R₁, R₂, R₃ and R₄, if present, are each independently            selected from the group consisting of hydrogen, C₁₋₅            alkyl(C═O)— and C₁₋₅ alkyl;        -   , in each instance, is a single or double bond,        -   wherein in each instance where it is a double bond, R₁, R₂,            R₃ or R₄ attached to the double bond-O is absent; when R₁ is            absent, R₅ is hydrogen, hydroxyl, C₁₋₅ alkyl or C₁₋₅ alkoxy;            when R₃ is absent, R₆ is hydrogen, hydroxyl, C₁₋₅ alkyl or            C₁₋₅ alkoxy;        -   wherein in each instance where it is a single bond, R₁, R₂,            R₃ or R₄ attached to the double bond-O is present; when R₁            is present, R₅ is hydrogen;    -   when R₃ is present, R₆ is hydrogen;        -   Q is —(CR_(c)R_(d))_(v)—;            -   wherein R_(c) and R_(d) are independently hydrogen or                C₁₋₅ alkyl; and v is an integer from 2 to 6;        -   p is an integer from 1 to 10;        -   A is

-   -   -   -   wherein, L is S, O or N; and

        -   G, in each instance, is independently, hydrogen, or is taken            together with L to form a nitric oxide donor or is absent;

        -   X is hydrogen, C₁₋₅ alkyl or is taken together with N to            form a nitric oxide donor;

        -   B is one hydrogen or —Y—Z, wherein Y is a spacer and Z is a            polymer or a terminus;

        -   D is —NR_(a)R_(b), wherein R_(a) and R_(b) are independently            selected from the group consisting of hydrogen, formyl, C₁₋₅            alkyl(C═O)—, C₁₋₅ alkyl and C₁₋₅ alkyl ester;

        -   or D is

-   -   2. The polyglucosamine of embodiment 1, wherein at least one of        the X and G is taken together with the atom on the        polyglucosamine to which it is bound to form a nitric oxide        donor.    -   3. The polygucosamine of embodiment 1, comprising the structural        unit:    -   4.

-   -   wherein,    -   m is an integer from 1 to 10,000.    -   5. The polyglucosamine of embodiment 1, wherein the nitric oxide        donor is taken together with the atom on the polyglucosamine to        which it is bound is selected from the group consisting of a        diazeniumdiolate, nitrosothiol, a nitrosamine, a hydroxyl        nitrosamine, a hydroxyl amine, a hydroxyurea, and combination        thereof.    -   5. The polyglucosamine of embodiment 4, wherein the nitric oxide        donor is diazeniumdiolate.    -   6. The polyglucosamine of embodiment 3, wherein in is an integer        from 1 to 50.    -   7. The polyglucosamine of embodiment 3, wherein m is an integer        from 1 to 10.    -   8. The polyglucosamine of embodiment 1, comprising at least one        structural unit:

-   -   wherein,        -   D is —NR_(a)R_(b), wherein R_(a) and R_(b) are independently            selected from the group consisting of hydrogen, formyl, C₁₋₅            alkyl(C═O)—, C₁₋₅ alkyl and C₁₋₅ alkyl ester.    -   9. The polyglucosamine of embodiment 8, wherein        -   , in each instance, is a single bond        -   R₁, R₂, R₃ and R₄, are each hydrogen, and        -   R₅ and R₆ are each hydrogen.    -   10. The polyglucosamine of embodiment 9, comprising at least one        structural unit:

-   -   11. The polyglucosamine of embodiment 10, wherein B is hydrogen.    -   12. The polyglucosamine of embodiment 11, wherein B is —Y—Z.    -   13. The polyglucosamine of embodiment 12, wherein B is —Y—Z,        wherein Z has the structure:

-   -   wherein j, in each instance, is an integer from 1 to 100.    -   14. The polyglucosamine of embodiment 12, wherein Y has the        structure:

-   -   -   wherein,        -   R_(p), R_(q), R_(s) and R_(t), in each instance, are            independently, hydrogen or hydroxyl; and        -   k is an integer from 1 to 20.

    -   15. The polyglucosamine of embodiment 1, comprising the        structural unit:

-   -   wherein,    -   D is

-   -   16. The polyglucosamine of embodiment 15, wherein        -   , in each instance, is a single bond, and        -   R₁, R₂, R₃ and R₄, are each hydrogen.    -   17. The polyglucosamine of embodiment 1, wherein        -   B is —Y—Z, wherein Z has the structure:

-   -   wherein j, in each instance, is an integer from 1 to 100.    -   18. The polyglucosamine of embodiment 17, wherein j is an        integer from 1 to 50.    -   19. The polyglucosamine of embodiment 17, wherein j is an        integer from 1 to 15.    -   20. The polyglucosamine of embodiment 1, wherein        -   A is

-   -   -   -   wherein G is hydrogen, or is taken together with N to                form a nitric oxide donor or is absent; and

        -   B is hydrogen.

    -   21. The polyglucosamine of embodiment 1, comprising the        structural unit:

-   -   -   wherein,        -   m is an integer from 1 to 1,000, and        -   n is an integer from 1 to 1,000.

    -   22. The polyglucosamine of embodiment 21, wherein m and n are        each independently selected from an integer of 1 to 50.

    -   23. The polyglucosamine of embodiment 21, comprising the        structural unit:

    -   24.

-   -   25. The polyglucosamine of embodiment 20, wherein        -   X is hydrogen or is taken together with N to form a            diazeniumdiolate; and        -   A is

-   -   -   -   wherein G is hydrogen or is taken together with N to                form a diazeniumdiolate.

    -   26. The polyglucosamine of embodiment 21, wherein        -   B is —Y—Z, wherein Z has the structure:

-   -   wherein j, in each instance, is an integer from 1 to 100.    -   27. The polyglucosamine of embodiment 22, wherein Y has the        structure:    -   28.

-   -   -   wherein,        -   R_(p), R_(q), R_(s) and R_(t), in each instance, are            independently, hydrogen or hydroxyl; and        -   k is an integer from 1 to 20.

    -   29. The polyglucosamine of embodiment 1, wherein A is N.

    -   30. The polyglucosamine of embodiment 1, wherein A is S.

    -   31. The polyglucosamine of embodiment 1, wherein        -   R_(c) and R_(d) are independently hydrogen or methyl; and        -   v is 2.

    -   32. A method of delivering nitric oxide to a subject,        comprising:        -   administering an effective amount of the polyglucosamine of            claim 1 to the subject.

    -   33. A method of treating a disease state, comprising:        -   administering an effective amount of the polyglucosamine of            embodiment 1 to a subject in need thereof, wherein the            disease state is selected from the group consisting of a            cancer, a cardiovascular disease, a microbial infection;            platelet aggregation and platelet adhesion caused by the            exposure of blood to a medical device; pathological            conditions resulting from abnormal cell proliferation;            transplantation rejections, autoimmune diseases,            inflammation, vascular diseases; scar tissue; wound            contraction, restenosis, pain, fever, gastrointestinal            disorders, respiratory disorders, sexual dysfunctions, and            sexually transmitted diseases.

    -   34. The method of embodiment 31, wherein said disease state is        cystic fibrosis.

    -   35. A pharmaceutical formulation comprising:        -   i. the polyglucosamine of claim 1; and        -   ii. a pharmaceutically acceptable carrier.

    -   36. The pharmaceutical formulation of embodiment 33, wherein the        polyglucosamine is water-soluble.

    -   35. The polyglucosamine of embodiment 1, wherein the        polyglucosamine is water soluble.

In all embodiments, combinations of substituents and/or variables arepermissible only if such combinations result in compounds that conformto a known valence for each atom.

Specific functionalized polyglucosamines include:

In each of the above structures of formula I and embodiments therein(i.e., Formulae III, VI and VII), when present, m is an integer from 1to 10,000, preferably 1 to 1000 or 1 to 500; more preferably 1 to 200 or1 to 100; and most preferably 1 to 50; 1 to 20 or 1 to 10, such as 1, 2,3, 4, 5, 6, 7, 8, 9 or 10; n is an integer from 1 to 10,000, preferably1 to 1000 or 1 to 500; more preferably 1 to 200 or 1 to 100; and mostpreferably 1 to 50; 1 to 20 or 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8,9 or 10; and 1 is an integer from 1 to 10,000, preferably 1 to 1000 or 1to 500; more preferably 1 to 200 or 1 to 100; and most preferably 1 to50; 1 to 20 or 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In an embodiment, the subject matter disclosed herein is directed to amethod of delivering nitric oxide to a subject, comprising administeringan effective amount of a functionalized polyglucosamine to a subject.

In another embodiment, the subject matter disclosed herein is directedto a method of treating a disease state, comprising administering aneffective amount of said polyglucosamine of claim 1 to a subject in needthereof, wherein said disease state is selected from the groupconsisting of a cancer, a cardiovascular disease, a microbial infection;platelet aggregation and platelet adhesion caused by the exposure ofblood to a medical device; pathological conditions resulting fromabnormal cell proliferation; transplantation rejections, autoimmunediseases, inflammation, vascular diseases; scar tissue; woundcontraction, restenosis, pain, fever, gastrointestinal disorders,respiratory disorders, sexual dysfunctions, and sexually transmitteddiseases. Preferably, the disease state is cystic fibrosis.

In another embodiment, the subject matter disclosed herein is directedto a method of disrupting, eradicating or preventing a biofilm. Thismethod comprises contacting a surface or area that contains a biofilm oris susceptible to a biofilm forming or occupying some or all of thesurface or area with a functionalized polyglucosamine as describedherein. The term “biofilm” is intended to mean an aggregate of one ormore microorganisms in which cells adhere to each other, usually on asurface. Most any free-floating microorganisms can form a biofilm and/orattach to a surface. Microorganisms can adhere to a surface or eachother through weak, reversible adhesion via van der Waals forces. Themicroorganisms can more permanently anchor using cell adhesion orstructures such as pili.

In yet another embodiment, the subject matter disclosed herein isdirected to a pharmaceutical formulation comprising a functionalizedpolyglucosamine and a pharmaceutically acceptable carrier. Preferably,the functionalized polyglucosamine is water-soluble as describedthroughout the present disclosure.

“Pharmaceutically acceptable,” as used herein, means a material that isnot biologically or otherwise undesirable, i.e., the material can beadministered to an individual along with the compositions of thisinvention, without causing substantial deleterious biological effects orinteracting in a deleterious manner with any of the other components ofthe composition in which it is contained. The material would naturallybe selected to minimize any degradation of the active ingredient and tominimize any adverse side effects in the subject, as would be well knownto one of skill in the art (see, e.g., Remington's PharmaceuticalScience; 20 ed. 2005). Exemplary pharmaceutically acceptable carriersfor the compositions of this invention include, but are not limited to,sterile pyrogen-free water and sterile pyrogen-free physiological salinesolution.

The presently disclosed therapeutic compositions, in some embodiments,comprise a composition that includes a presently disclosed nitricoxide-releasing polyglucosamine and a pharmaceutically acceptablecarrier. Suitable compositions include aqueous and non-aqueous sterileinjection solutions that can contain antioxidants, buffers,bacteriostats, bactericidal antibiotics and solutes that render theformulation isotonic with the bodily fluids of the intended recipient;and aqueous and non-aqueous sterile suspensions, which can includesuspending agents and thickening agents.

The compositions used in the presently disclosed methods can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents, such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The therapeutic compositions can be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and can be stored ina frozen or freeze-dried (lyophilized) condition requiring only theaddition of sterile liquid carrier immediately prior to use.

For oral administration, the compositions can take the form of, forexample, tablets or capsules prepared by a conventional technique withpharmaceutically acceptable excipients, such as binding agents (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods known in the art. For example, atherapeutic agent can be formulated in combination withhydrochlorothiazide, and as a pH stabilized core having an enteric ordelayed release coating which protects the therapeutic agent until itreaches the target organ.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional techniqueswith pharmaceutically acceptable additives, such as suspending agents(e.g., sorbitol syrup, cellulose derivatives or hydrogenated ediblefats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations also cancontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration can be suitablyformulated to give controlled release of the active compound. For buccaladministration the compositions can take the form of tablets or lozengesformulated in conventional manner.

The compounds also can be formulated as a preparation for implantationor injection. Thus, for example, the compounds can be formulated withsuitable polymeric or hydrophobic materials (e.g., as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives (e.g., as a sparingly soluble salt). The compounds also canbe formulated in rectal compositions (e.g., suppositories or retentionenemas containing conventional suppository bases, such as cocoa butteror other glycerides), creams or lotions, or transdermal patches.

Pharmaceutical formulations also are provided which are suitable foradministration as an aerosol by inhalation. Preferably, thefunctionalized polyglucosamines described herein are formulated insolution and/or aerosol form. These formulations comprise a solution orsuspension of a NO-releasing polyglucosamine described herein. Thedesired formulation can be placed in a small chamber and nebulized.Nebulization can be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the NO-releasing polyglucosamine. For example, the presentlydisclosed NO-releasing polyglucosamine can be administered viainhalation to treat bacterial infections related to cystic fibrosis.Cystic fibrosis-related bacterial infections include, but are notlimited to stenotrophomonis, Mybacterium avium intracellulaire and M.abcessus, Burkhoderia cepacia and Pseudomonas aeruginosa (P. aeruginosa)infections.

The term “effective amount” is used herein to refer to an amount of thetherapeutic composition (e.g., a composition comprising a nitricoxide-releasing polyglucosamine) sufficient to produce a measurablebiological response. Actual dosage levels of active ingredients in anactive composition of the presently disclosed subject matter can bevaried so as to administer an amount of the active compound(s) that iseffective to achieve the desired response for a particular subjectand/or application. The selected dosage level will depend upon a varietyof factors including the activity of the composition, formulation, theroute of administration, combination with other drugs or treatments,severity of the condition being treated, and the physical condition andprior medical history of the subject being treated. Preferably, aminimal dose is administered, and dose is escalated in the absence ofdose-limiting toxicity to a minimally effective amount. Determinationand adjustment of an effective dose, as well as evaluation of when andhow to make such adjustments, are known to those of ordinary skill inthe art of medicine.

For administration of a composition as disclosed herein, conventionalmethods of extrapolating human dosage based on doses administered to amurine animal model can be carried out using the conversion factor forconverting the mouse dosage to human dosage: Dose Human per kg=DoseMouse per kg×12. See Freireich et al., Cancer Chemother Rep. 50, 219-244(1966). Drug doses also can be given in milligrams per square meter ofbody surface area because this method rather than body weight achieves agood correlation to certain metabolic and excretionary functions.Moreover, body surface area can be used as a common denominator for drugdosage in adults and children as well as in different animal species.See Freireich et al., Cancer Chemother Rep. 50, 219-244 (1966). Briefly,to express a mg/kg dose in any given species as the equivalent mg/sq mdose, multiply the dose by the appropriate km factor. In an adult human,100 mg/kg is equivalent to 100 mg/kg×37 kg/sq m=3700 mg/m².

For additional guidance regarding formulation and dose, see U.S. Pat.Nos. 5,326,902; 5,234,933; PCT International Publication No. WO93/25521; Berkow et al., The Merck Manual of Medical Information, Homeed., Merck Research Laboratories: Whitehouse Station, N.J. (1997);Goodman et al., Goodman & Gilman's the Pharmacological Basis ofTherapeutics, 9th ed. McGraw-Hill Health Professions Division: New York(1996); Ebadi, CRC Desk Reference of Clinical Pharmacology, CRC Press,Boca Raton, Fla. (1998); Katzunq, Basic & Clinical Pharmacology, 8th ed.Lange Medical Books/McGraw-Hill Medical Pub. Division: New York (2001);Remington et al., Remington's Pharmaceutical Sciences, 15th ed. MackPub. Co.: Easton, Pa. (1975); and Speight et al., Avery's DrugTreatment: A Guide to the Properties, Choice, Therapeutic Use andEconomic Value of Drugs in Disease Management, 4th ed. AdisInternational: Auckland/Philadelphia (1997); Dutch et al., Toxicol.Leu., 100-101, 255-263 (1998).

Suitable methods for administering to a subject a composition of thepresently disclosed subject matter include, but are not limited to,systemic administration, parenteral administration (includingintravascular, intramuscular, intraarterial administration), oraldelivery, buccal delivery, subcutaneous administration, inhalation,intratracheal installation, surgical implantation, transdermal delivery,local injection, and hyper-velocity injection/bombardment. Whereapplicable, continuous infusion can enhance drug accumulation at atarget site (see, e.g., U.S. Pat. No. 6,180,082).

The particular mode of drug administration used in accordance with themethods of the presently disclosed subject matter depends on variousfactors, including but not limited to the agent and/or carrier employed,the severity of the condition to be treated, and mechanisms formetabolism or removal of the active agent following administration.

In some embodiments, one or more additional therapeutic agents can beused in combination with the functionalized polyglucosamine. Suchadditional agents can be part of a formulation comprising thefunctionalized polyglucosamine or dosed as a separate formulation priorto, after, or at the same time (concurrently) as a formulation includingthe functionalized polyglucosamine. Such additional therapeutic agentsinclude, in particular, anti-cancer therapeutics, anti-microbial agents,pain relievers, anti-inflammatories, vasodialators, andimmune-suppressants, as well as any other known therapeutic agent thatcould enhance the alleviation of the disease or condition being treated.“Concurrently” means sufficiently close in time to produce a combinedeffect (that is, concurrently can be simultaneously, or it can be two ormore events occurring within a short time period before or after eachother). In some embodiments, the administration of two or more compounds“concurrently” means that the two compounds are administered closelyenough in time that the presence of one alters the biological effects ofthe other. The two compounds can be administered in the same ordifferent formulations or sequentially. Concurrent administration can becarried out by mixing the compounds prior to administration, or byadministering the compounds in two different formulations, for example,at the same point in time but at different anatomic sites or usingdifferent routes of administration.

The choice of additional therapeutic agents to be used in combinationwith an NO-releasing polyglucosamine will depend on various factorsincluding, but not limited to, the type of disease, the age, and thegeneral health of the subject, the aggressiveness of diseaseprogression, and the ability of the subject to tolerate the agents thatcomprise the combination.

A variety of chemical compounds, also described as “antineoplastic”agents or “chemotherapeutic agents” can be used in combination with thepresently disclosed NO-releasing polyglucosamines used in the treatmentof cancer. Such chemotherapeutic compounds include, but are not limitedto, alkylating agents, DNA intercalators, protein synthesis inhibitors,inhibitors of DNA or RNA synthesis, DNA base analogs, topoisomeraseinhibitors, anti-angiogenesis agents, and telomerase inhibitors ortelomeric DNA binding compounds. For example, suitable alkylating agentsinclude alkyl sulfonates, such as busulfan, improsulfan, and piposulfan;aziridines, such as a benzodizepa, carboquone, meturedepa, and uredepa;ethylenimines and methylmelamines, such as altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, cyclophosphamide, estramustine,iphosphamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichine, phenesterine, prednimustine, trofosfamide, anduracil mustard; nitroso ureas, such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, and ranimustine.

Antibiotics used in the treatment of cancer include dactinomycin,daunorubicin, doxorubicin, idarubicin, bleomycin sulfate, mytomycin,plicamycin, and streptozocin. Chemotherapeutic antimetabolites includemercaptopurine, thioguanine, cladribine, fludarabine phosphate,fluorouracil (5-FU), floxuridine, cytarabine, pentostatin, methotrexate,and azathioprine, acyclovir, adenine β-1-D-arabinoside, amethopterin,aminopterin, 2-aminopurine, aphidicolin, 8-azaguanine, azaserine,6-azauracil, 2′-azido-2-deoxynucleotides, 5-bromodeoxycytidine, cytosineβ-1-D-arabinoside, diazooxonorleucine, dideoxynucleosides,5-fluorodeoxycytidine, 5-fluorodeoxyuridine, and hydroxyurea.

Chemotherapeutic protein synthesis inhibitors include abrin,aurintricarboxylic acid, chloramphenicol, colicin E3, cycloheximide,diphtheria toxin, edeine A, emetine, erythromycin, ethionine, fluoride,5-fluorotryptophan, fusidic acid, guanylyl methylene diphosphonate andguanylyl imidodiphosphate, kanamycin, kasugamycin, kirromycin, andO-methyl threonine. Additional protein synthesis inhibitors includemodeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin,ricin, shiga toxin, showdomycin, sparsomycin, spectinomycin,streptomycin, tetracycline, thiostrepton, and trimethoprim. Inhibitorsof DNA synthesis, including alkylating agents such as dimethyl sulfate,mitomycin C, nitrogen and sulfur mustards, intercalating agents, such asacridine dyes, actinomycins, adriamycin, anthracenes, benzopyrene,ethidium bromide, propidium diiodide-intertwining, and agents, such asdistamycin and netropsin, can be used as part of the presently disclosedcancer treatments. Topoisomerase inhibitors, such as coumermycin,nalidixic acid, novobiocin, and oxolinic acid, inhibitors of celldivision, including colcemide, colchicine, vinblastine, and vincristine;and RNA synthesis inhibitors including actinomycin D, a-amanitine andother fungal amatoxins, cordycepin (3′-deoxyadenosine),dichlororibofuranosyl benzimidazole, rifampicine, streptovaricin, andstreptolydigin also can be combined with functionalized polyglucosaminesto provide a suitable cancer treatment.

Thus, current chemotherapeutic agents that can be used in combinationwith the presently described NO-releasing functionalizedpolyglucosamines include, adrimycin, 5-fluorouracil (5FU), etoposide,camptothecin, actinomycin-D, mitomycin, cisplatin, hydrogen peroxide,carboplatin, procarbazine, mechlorethamine, cyclophosphamide,ifosfamide, melphalan, chjlorambucil, bisulfan, nitrosurea,dactinomycin, duanorubicin, doxorubicin, bleomycin, pilcomycin,tamoxifen, taxol, transplatimun, vinblastin, and methotrexate, and thelike.

As used herein, the term “antimicrobial agent” refers to any agent thatkills, inhibits the growth of, or prevents the growth of a bacteria,fungus, yeast, or virus. Suitable antimicrobial agents that can beincorporated into the presently disclosed NO-releasing functionalizedpolyglucosamines to aid in the treatment or prevention of a microbialinfection, include, but are not limited to, antibiotics such asvancomycin, bleomycin, pentostatin, mitoxantrone, mitomycin,dactinomycin, plicamycin and amikacin. Other antimicrobial agentsinclude antibacterial agents such as 2-p-sulfanilyanilinoethanol,4,4′-sulfinyldianiline, 4-sulfanilamidosalicylic acid, acediasulfone,acetosulfone, amikacin, amoxicillin, amphotericin B, ampicillin,apalcillin, apicycline, apramycin, arbekacin, aspoxicillin,azidamfenicol, azithromycin, aztreonam, bacitracin, bambermycin(s),biapenem, brodimoprim, butirosin, capreomycin, carbenicillin,carbomycin, carumonam, cefadroxil, cefamandole, cefatrizine,cefbuperazone, cefclidin, cefdinir, cefditoren, cefepime, cefetamet,cefixime, cefmenoxime, cefininox, cefodizime, cefonicid, cefoperazone,ceforanide, cefotaxime, cefotetan, cefotiam, cefozopran, cefpimizole,cefpiramide, cefpirome, cefprozil, cefroxadine, ceftazidime, cefteram,ceftibuten, ceftriaxone, cefuzonam, cephalexin, cephaloglycin,cephalosporin C, cephradine, chloramphenicol, chlortetracycline,ciprofloxacin, clarithromycin, clinafloxacin, clindamycin, clindamycinphosphate, clomocycline, colistin, cyclacillin, dapsone, demecicycline,diathymosulfone, dibekacin, dihydrostreptomycin, dirithromycin,doxycycline, enoxacin, enviomycin, epicillin, erythromycin, flomoxef,fortimicin(s), gentamicin(s), glucosulfone solasulfone, gramicidin S,gramicidin(s), grepafloxacin, guamecycline, hetacillin, imipenem,isepamicin, josamycin, kanamycin(s), leucomycin(s), lincomycin,lomefloxacin, lucensomycin, lymecycline, meclocycline, meropenem,methacycline, micronomicin, midecamycin(s), minocycline, moxalactam,mupirocin, nadifloxacin, natamycin, neomycin, netilmicin, norfloxacin,oleandomycin, oxytetracycline, p-sulfanilylbenzylamine, panipenem,paromomycin, pazufloxacin, penicillin N, pipacycline, pipemidic acid,polymyxin, primycin, quinacillin, ribostamycin, rifamide, rifampin,rifamycin SV, rifapentine, rifaximin, ristocetin, ritipenem,rokitamycin, rolitetracycline, rosaramycin, roxithromycin,salazosulfadimidine, sancycline, sisomicin, sparfloxacin, spectinomycin,spiramycin, streptomycin, succisulfone, sulfachrysoidine, sulfaloxicacid, sulfamidochrysoidine, sulfanilic acid, sulfoxone, teicoplanin,temafloxacin, temocillin, tetracycline, tetroxoprim, thiamphenicol,thiazolsulfone, thiostrepton, ticarcillin, tigemonam, tobramycin,tosufloxacin, trimethoprim, trospectomycin, trovafloxacin,tuberactinomycin and vancomycin. Antimicrobial agents can also includeanti-fungals, such as amphotericin B, azaserine, candicidin(s),chlorphenesin, dermostatin(s), filipin, fungichromin, mepartricin,nystatin, oligomycin(s), perimycin A, tubercidin, imidazoles, triazoles,and griesofulvin.

In some embodiments, the NO-releasing polyglucosamine can beincorporated into polymeric films. Such incorporation can be throughphysically embedding the polyglucosamine into polymer surfaces, viaelectrostatic association of the polyglucosamine onto polymericsurfaces, or by covalent attachment of functionalized polyglucosamineonto reactive groups on the surface of a polymer. Alternatively, thefunctionalized polyglucosamine can be mixed into a solution of liquidpolymer precursor, becoming entrapped in the polymer matrix when thepolymer is cured. Polymerizable groups can also be used to furtherfunctionalize the functionalized polyglucosamine, whereupon, thepolyglucosamine can be co-polymerized into a polymer during thepolymerization process. Suitable polymers into which the NO-releasingpolyglucosamine can be incorporated include polyolefins, such aspolystyrene, polypropylene, polyethylene, polytetrafluoroethylene, andpolyvinylidene, as well as polyesters, polyethers, polyurethanes, andthe like. In particular, polyurethanes can include medically segmentedpolyurethanes. Medically segmented polyurethanes can also include one ormore expander moieties, such as alkylene chains, that add additionallength or weight to the polymer. Such polyurethanes are also generallynon-toxic. One example of a medically segmented polyurethane isTECOFLEX®.

Polymeric films containing NO-releasing polyglucosamines can be used tocoat a variety of articles, particularly surgical tools, biologicalsensors, and medical implants to prevent platelet adhesion, to preventbacterial infection, to act as a vasodilator. These articles can be ofuse in vascular medical devices, urological medical devised, biliarymedical devices, gastrointestinal medical devices, medical devicesadapted for placement at surgical sites, and medical devices adapted forplacement on skin wounds or openings. Thus, the polymers can be used tocoat arterial stents, guide wires, catheters, trocar needles, boneanchors, bone screws, protective platings, hip and joint replacements,electrical leads, biosensors, probes, sutures, surgical drapes, wounddressings, and bandages.

In some embodiments, the device being coated can have a metallicsurface, such as, for example, stainless steel, nickel, titanium,aluminum, copper, gold, silver, platinum, and combinations thereof. Insome embodiments, the films or polymers containing the NO-releasingpolyglucosamine can be used to coat non-metallic surfaces, such as glassor fiber (e.g., cloth or paper).

Additionally, polymers containing NO-releasing polyglucosamine can beused to form the devices, themselves. For example, the polymers can befashioned into storage bags for blood or tissue or as wound dressings.

Surfaces that can be contacted with a functionalized polyglucosamine toprevent or disrupt biofilms include those selected from the groupconsisting of medical devices, plumbing fixtures, condenser coils,optical surfaces, boat hulls and aircrafts. Other non-limiting examplesinclude counter tops, windows, appliances, hard floors, rugs, tubs,showers, mirrors, toilets, bidets, bathroom fixtures, sinks,refrigerators, microwaves, small kitchen appliances, tables, chairs,cabinets, drawers, sofas, love seats, benches, beds, stools, armoires,chests, dressers, display cabinets, clocks, buffets, shades, shutters,entertainment centers, arm rails, lamps, banisters, libraries, cabinets,desks, doors, shelves, couches, carts, pianos, statues and other art,racks, fans, light fixtures, pool tables, ping pong tables, soccertables, card tables, tools (e.g., hand powered and/or hand held tools,electrical tools, air powered tools, etc.), telephones, radios,televisions, stereo equipment, CD and DVD players, analog and digitalsound devices, palm computers, laptop computers, desktop and towercomputers, computer monitors, mp3 players, memory storage devices,cameras, camcorders, vehicle surfaces (e.g., windshield; tires; metal,fiberglass, composite material and/or plastic outer surfaces; fabricand/or vinyl outer surfaces; fabric, vinyl, and/or leather interiorsurfaces; metal, plastic, wood and/or composite material interiorsurfaces, glass interior surfaces, etc.), bicycles, snowmobiles,motorcycles, off-road-vehicles, yard equipment, farm equipment, washingequipment (e.g., power washers, etc.), painting equipment (e.g.,electric and air powered painting equipment, etc.), medical and/ordental equipment, marine equipment (e.g., sail boats, power boats,rafts, sail board, canoe, row boats, etc.), toys, writing implements,watches, framed pictures or paintings, books, and/or the like. Anysurface where it is desirable to cause one or more types of liquids torun off of a surface, to not be absorbed into a surface, and/or to notstain a surface, can be a substrate. For example, a surface that isexposed to environmental conditions. Also where the surface can become alocus for microbial adhesion such as medical devices that contact bodilytissues or fluids is particularly preferred.

Medical devices such as catheters, which are adapted for movementthrough blood vessels or other body lumens, are typically provided withlow-friction outer surfaces. If the surfaces of the medical devices arenot low-friction surfaces, insertion of the devices into and removal ofthe devices from the body lumens becomes more difficult, and injury orinflammation of bodily tissue may occur. Low friction surfaces are alsobeneficial for reducing discomfort and injury that may arise as a resultof movement between certain long term devices (e.g., long termcatheters) and the surrounding tissue, for example, as a result ofpatient activity. Medical devices include a variety of implantable andinsertable medical devices (also referred to herein as “internal medicaldevices”). Examples of such medical devices include, devices involvingthe delivery or removal of fluids (e.g., drug containing fluids,pressurized fluids such as inflation fluids, bodily fluids, contrastmedia, hot or cold media, etc.) as well as devices for insertion intoand/or through a wide range of body lumens, including lumens of thecardiovascular system such as the heart, arteries (e.g., coronary,femoral, aorta, iliac, carotid and vertebro-basilar arteries) and veins,lumens of the genitourinary system such as the urethra (includingprostatic urethra), bladder, ureters, vagina, uterus, spermatic andfallopian tubes, the nasolacrimal duct, the eustachian tube, lumens ofthe respiratory tract such as the trachea, bronchi, nasal passages andsinuses, lumens of the gastrointestinal tract such as the esophagus,gut, duodenum, small intestine, large intestine, rectum, biliary andpancreatic duct systems, lumens of the lymphatic system, the major bodycavities (peritoneal, pleural, pericardial) and so forth. Non-limiting,specific examples of internal medical devices include vascular devicessuch as vascular catheters (e.g., balloon catheters), including balloonsand inflation tubing for the same, hydrolyses catheters, guide wires,pullback sheaths, filters (e.g., vena cava filters), left ventricularassist devices, total artificial hearts, injection needles, drugdelivery tubing, drainage tubing, gastroenteric and colonoscopic tubing,endoscopic devices, endotracheal devices such as airway tubes, devicesfor the urinary tract such as urinary catheters and ureteral stents, anddevices for the neural region such as catheters and wires, trocarneedles, bone anchors, bone screws, protective platings, jointreplacements, electrical leads, biosensors, probes, sutures, surgicaldrapes, wound dressings, and bandages. Many devices in accordance withthe invention have one or more portions that are cylindrical in shape,including both solid and hollow cylindrical shapes.

Solid substrate materials can include organic materials (e.g., materialscontaining 50 wt % or more organic species) such as polymeric materials,and inorganic materials (e.g., materials containing 50 wt % or moreinorganic species), such as metallic materials (e.g., metals and metalalloys) and non-metallic materials (e.g., including carbon,semiconductors, glasses and ceramics, which may contain various metal-and non-metal-oxides, various metal- and non-metal-nitrides, variousmetal- and non-metal-carbides, various metal- and non-metal-borides,various metal- and non-metal-phosphates, and various metal- andnon-metal-sulfides, among others). Specific examples of non-metallicinorganic materials can be materials containing one or more of thefollowing: metal oxides, including aluminum oxides and transition metaloxides (e.g., oxides of titanium, zirconium, hafnium, tantalum,molybdenum, tungsten, rhenium, and iridium); silicon; silicon-basedceramics, such as those containing silicon nitrides, silicon carbidesand silicon oxides (sometimes referred to as glass ceramics); calciumphosphate ceramics (e.g., hydroxyapatite); carbon; and carbon-based,ceramic-like materials such as carbon nitrides.

Further, the NO-releasing polyglucosamine can be incorporated intodetergents, such as, but not limited to, anti-microbial soaps. Forexample, NO-release from functionalized polyglucosamine embedded in barsoaps can be triggered by contact with water and/or a drop in pH uponuse. As the outer surface of the bar is eroded or dissolved, additionalfunctionalized polyglucosamine within the bar surface become exposed forsubsequent uses of the bar. NO-releasing polyglucosamine also can besuspended in liquid soaps. Such soaps or detergents can be used forpersonal hygiene or to provide anti-microbial treatments for fibers.Such soaps or detergents can also be used to treat household surfaces orany surface in a hospital or other medical environment that may beexposed to microbes such as bacteria, fungi or viruses.

The term “biocompatible” refers herein to organic solvents that do notinduce toxic or unwanted side effects when administered to a patient incertain amounts.

The formulations include all pharmaceutically acceptable salt formsthereof. Examples of such salts include those derived frompharmaceutically acceptable inorganic and organic acids and bases.Examples of suitable acid salts include, without limitation, acetate,adipate, alginate, aspartate, benzoate, butyrate, citrate, fumarate,glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,malonate, methanesulfonate, nicotinate, nitrate, oxalate, palmoate,pectinate, persulfate, hydroxynaphthoate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate andundecanoate. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, can be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

Salts derived from appropriate bases include, without limitation, alkalimetal (e.g., sodium, potassium), alkaline earth metal (e.g., magnesiumand calcium), ammonium and N-(alkyl)₄ ⁺ salts.

The functionalized polyglucosamines also include those havingquaternization of any basic nitrogen-containing group therein.

The discussion herein is, for simplicity, provided without reference tostereoisomerism. Those skilled in the art will appreciate that thepolyglucosamines described herein can contain one or more asymmetriccenters and thus occur as racemates and racemic mixtures, single opticalisomers, individual diastereomers, and diastereomeric mixtures. All suchisomeric forms of these compounds are expressly included in the presentinvention.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES

1. Synthesis of NO-Releasing Chitosan Oligosaccharides

Chitosan oligosaccharides were synthesized by the oxidation (hydrogenperoxide) (Kim, S. K.; Rajapakse, N. Enzymatic production and biologicalactivities of chitosan oligosaccharides (COS): A review. Carbohydr.Polym. 2005, 62, 357-368) or enzymatic degradation of chitosanpolysaccharides. The resulting water soluble chitosan oligosaccharideswere modified by a cationic ring opening of aziridine compounds(including but not limited to aziridine and 2-methyl aziridine) toimpart secondary amine moieties, followed by the reaction with highpressure NO under basic condition (“charging”) to yield water solubleNO-releasing chitosan oligosaccharides.

Materials and Methods

Medium molecular weight chitosan, 2-methyl aziridine (MAz), rhodamine Bisothiocyanate (RITC), poly(ethylene glycol) methyl ether acrylate(average Mn=480) (PEG), fetal bovine serum (FBS), Dulbecco's ModifiedEagle's Medium (DMEM), phenazine methosulfate (PMS),3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliuminner salt (MTS), trypsin, phosphate buffered saline (PBS), andpenicillin streptomycin (PS) were purchased from the Aldrich ChemicalCompany (Milwaukee, Wis.). Pseudomonas aeruginosa (ATCC #19143) wasobtained from the American Type Culture Collection (Manassas, Va.).Trypic soy broth (TSB) and Tryptic soy agar (TSA) are purchased fromBecton, Dickinson, and Company (Franklin Lakes, N.J.). L929 mousefibroblasts (ATCC # CCL-1) were obtained from the University of NorthCarolina Tissue Culture Facility (Chapel Hill, N.C.). Distilled waterwas purified with a Millipore Milli-Q Gradient A-10 water purificationsystem (Bedford, Mass.). Syto 9 green fluorescent nucleic acid stain waspurchased from Life Technologies (Grand Island, N.Y.). Common laboratorysalts and solvents were purchased from Fisher Scientific (Pittsburgh,Pa.). All materials were used as received without further purificationunless noted otherwise. Nuclear magnetic resonance (NMR) spectra wererecorded on a 400 MHz Bruker instrument. Elemental (carbon, hydrogen,and nitrogen or CHN) analysis was performed using a PerkinElmerElemental Analyzer Series 2400 instrument (Waltham, Mass.).

Chitosan oligosaccharides were prepared by oxidative degradation usinghydrogen peroxide. Medium molecular weight chitosan (2.5 g) wassuspended in a hydrogen peroxide solution (15 or 30 wt %) under stirringfor 1 h at 65-85° C. Following the removal of undissolved chitosan byfiltration, chitosan oligosaccharides were precipitated from solution byadding acetone to the filtrate. The precipitate was collected bycentrifugation, washed twice with ethanol, and dried under vacuum atroom temperature. The viscosity of the chitosan oligosaccharides wasmeasured in a solution of NaCl (0.20 M) and CH3COOH (0.10 M) at 25° C.using an Ubbleohde capillary viscometer. Oligosaccharide molecularweight was determined using the classic Mark-Houwink equation (i.e.,[η]=1.81×10−3 M^(0.93)). Maghami (1988).

Control of the molecular weight (M_(w)) was achieved by varying theconcentration of hydrogen peroxide and degradation temperature. Theviscosity of the chitosan oligosaccharides was determined in a solutionof sodium chloride (0.20 M) and acetic acid (0.10 M) using an Ubbleohdecapillary viscometer. Du, J.; Hsieh, Y. L. Nanofibrous membranes fromaqueous electrospinning of carboxymethyl chitosan. Nanotechnology 2008,19, 125707. In combination with the Mark-Houwink equation (i.e.,[η]=1.81×10⁻³ M^(0.93)), molecular weights were determined as a functionof processing conditions. Collectively, larger concentrations ofhydrogen peroxide and elevated degradation temperatures led to lowermolecular weight chitosan. As shown in Table 1, chitosanoligosaccharides of ˜10 kD molecular weight were prepared in 15 wt %hydrogen peroxide at 65° C. for 1 h. Increasing the degradationtemperature to 85° C. resulted in significantly smaller size (MW˜5 kD).

TABLE 1 Table 1 Degradation conditions and elemental analysis ofchitosan oligosaccharides of different molecular weights. Chitosanoligosaccharides M_(v) ^(a) T (° C.) [H₂O₂] (wt %)   2.5k 2657 85 30 5k5370 85 15 10k  10142 65 15 ^(a)viscosity average molecular weight asdetermined by classic Mark-Houwink equation (i.e., [η] = 1.81 × 10⁻³M^(0.93)).

When both a larger concentration of hydrogen peroxide (i.e., 30 wt %)and elevated temperature (i.e., 85° C.) were adopted, the molecularweight of chitosan oligosaccharides decreased further (˜2.5 kD) wereachieved. As shown in Table 2, the CHN elemental analysis of theoligosaccharides indicated an overall nitrogen content of 6.3 wt %.

TABLE 2 Elemental (CHN) analysis of chitosan oligosaccharides andsecondary amine-functionalized derivatives. Materials C (%) H (%) N (%)Chitosan oligosaccharides^(a) 42.2 ± 1.6 6.8 ± 0.1  6.3 ± 0.2 Chitosan1-5k 43.5 ± 1.2 7.7 ± 0.3  8.9 ± 0.1 Chitosan 2-5k 44.7 ± 1.8 8.4 ± 0.210.8 ± 0.8 Chitosan 3-5k 51.0 ± 0.2 9.0 ± 0.2  3.1 ± 0.1 Chitosan 2-2.5k43.7 ± 0.7 8.5 ± 0.2 10.9 ± 0.1 Chitosan 2-10k 44.7 ± 1.8 8.4 ± 0.2 10.8± 0.8 ^(a)chitosan oligosaccharides before the grafting of 2-methylaziridine. Each parameter was analyzed with multiple replicates (n = 3).

2-methyl aziridine (MAz) was grafted onto the chitosan oligosaccharidesat different feed ratios (i.e., 2:1 and 1:1) to alter the secondaryamine functionalization and NO storage. Increasing the feed ratio of2-methyl aziridine to primary amines from 1:1 (CSO 1; Chitosan 1-5k) to2:1 (CSO 2; Chitosan 2-5k) resulted in greater NO storage (e.g., ˜0.30to 0.87 μmol/mg, respectively). As shown in FIGS. 5A & B, the NO fluxand storage of Chitosan 1/NO-5k were lower than Chitosan 2/NO-5k, aresult that may be attributable to the smaller amine concentration ofChitosan 1-5k (˜8.9 wt %) compared to Chitosan 2-5k (˜10.8 wt %).

Schemes A and B depict routes for preparing NO-releasing chitosanoligosaccharides described herein.

In Scheme A, CSO 1 (x=1); CSO 2 (x=2); CSO 1-NO (x=1); CSO 2-NO (x=2).In scheme B, CSO 3 (x=2); CSO 3-NO (x=2). Scheme A above is a syntheticroute for preparing secondary amine- and diazeniumdiolate-functionalizedchitosan oligosaccharides derivatives involves grafting of 2-methylaziridine onto primary amines of chitosan oligosaccharides (CSO 1, 2)and diazeniumdiolation of the resulting materials (CSO 1, 2-NO). SchemeB above is a synthetic route for preparing secondary amine- anddiazeniumdiolate-functionalized chitosan oligosaccharides derivativesinvolves PEGylation of 2-methyl aziridine-grafted-chitosanoligosaccharide (CSO 3) and the diazeniumdiolation of the resultingmaterial (CSO 3-NO).

Schemes A′ and B′ depict a synthesis route for chitosan oligosaccharidesdisclosed herein:

Reaction of the secondary amine-functionalized chitosan oligosaccharides(Chitosan 1, Chitosan 2, and Chitosan 3) with NO (10 atm under basicconditions) yielded N-diazeniumdiolate NO donor-functionalized chitosanoligosaccharides (Chitosan 1/NO, Chitosan 2/NO, and Chitosan 3/NO). TheNO conjugation (“charging”) solvent affects the charging efficiency andpotentially total NO storage. Carpenter, A. W.; Slomberg, D. L.; Rao, K.S.; Schoenfisch, M. H., Influence of scaffold size on bactericidalactivity of nitric oxide-releasing silica nanoparticles. ACS Nano 2012,5, 7235-7244. Aqueous solutions were necessary in order to adequatelydissolve the chitosan oligosaccharides. To examine the influence ofwater concentration on N-diazeniumdiolate conversion efficiency,mixtures of methanol (a common charging solvent) (Carpenter (2012);Stasko, N. A.; Schoenfisch, M. H. Dendrimers as a scaffold for nitricoxide release. J. Am. Chem. Soc. 2006, 128, 8265-8271) and water wereprepared (10:0, 9:1, 8:2, 7:3, and 6:4 v/v) and the pH was adjusted toabove 10 by adding sodium methoxide.

Scheme C depicts a route for preparing nitrosothiol NO-releasingchitosan oligosaccharides as described herein.

Scheme D depicts diazeniumdiolate conjugation and release of 2 NO from aconjugated diazeniumdiolate.

Secondary Amine-Functionalized Chitosan Oligosaccharides: 2-methylaziridine (MAz) grafted chitosan oligosaccharides were synthesizedfollowing a previously reported procedure. Wong, K.; Sun, G. B.; Zhang,X. Q.; Dai, H.; Liu, Y.; He, C. B.; Leong, K. W. PEI-g-chitosan, a novelgene delivery system with transfection efficiency comparable topolyethylenimine in vitro and after liver administration in vivo.Bioconj. Chem. 2006, 17, 152-158. Briefly, a mixture of concentrated HCl(11 μL), water (100 μL) and MAz with a 1:1 (CSO 1; Chitosan 1) or 2:1(CSO 2; Chitosan 2) molar ratio to primary amines on the chitosanoligosaccharides was added dropwise to a solution of chitosanoligosaccharides (100 mg) in deionized water (5 mL). The resultingsolution was stirred at room temperature for 5 d, and then at 75° C. for24 h. The product was purified by dialysis and collected bylyophilization. Any high molecular weight poly(2-methyl aziridine) inthe product was removed by washing with methanol, and the resultingmaterial was dried under vacuum at room temperature. Chitosan 2 was thendissolved in water at pH 10.0. The primary amine on the chitosanoligosaccharides was functionalized by adding poly(ethylene glycol)methyl ether acrylate to generate Chitosan 3. The resultingPEG-functionalized chitosan oligosaccharide derivative was purified bydialysis and collected by lyophilization. ₁H NMR data of Chitosan 1 andChitosan 2 (400 MHz, CD₃OD, δ): 0.8-1.1 (NH₂CH(CH₃)CH₂NH), 1.9 (C7:CHNHCOCH₃), 2.3-2.7 (NH₂CH(CH₃)CH₂NHCH, C2: NH₂CH(CH₃)CH₂NHCH), 3.3-4.0(C3, C4, C5, C6: OHCH, OCHCH(OH)CH(NH₂)CH, OHCH₂CH, OHCH₂CH), 4.4 (C1:OCH(CHNH₂)O). ₁H NMR data of Chitosan 3 (400 MHz, CD₃OD, δ): 0.8-1.1(NH₂CH(CH₃)CH₂NH), 1.9 (C7: CHNHCOCH₃), 2.3-2.7 (NH₂CH(CH₃)CH₂NHCH, C2:NH₂CH(CH₃)CH₂NHCH), 3.2 (OCH₂CH₂OCH₃), 3.3-4.0 (OCH₂CH₂O and C3, C4, C5,C6: OHCH, OCHCH(OH)CH(NH₂)CH, OHCH₂CH, OHCH₂CH), 4.4 (C1: OCH(CHNH₂)O).

N-Diazeniumdiolate-Functionalized Chitosan Oligosaccharides: Secondaryamine-functionalized chitosan oligosaccharides (CSO 1, Chitosan 1, CSO2, Chitosan 2, chitosan 3 and CSO 3) and 5.4 mM sodium methoxide (75 μL)were added to a methanol/water mixture (2 mL) of different v/v ratios(e.g., 10:0, 9:1, 8:2, 7:3, 6:4). The suspension was added to vials in aParr hydrogenation vessel, which was purged rapidly (5-10 s) with argonthree times followed by three longer argon purge cycles (10 min) toremove residual oxygen from the solution. The Parr hydrogenation vesselwas then pressurized to 10 atm with NO gas purified over KOH pellets (toremove NO degradation products) and maintained at 10 atm for 3 d. Thesame argon purging protocol was performed to remove unreacted NO anddegradation products from the solution prior to removing the vials fromthe vessel.

Fluorescently-Labeled Chitosan Oligosaccharides: These chitosanoligosaccharides were prepared following a previously reportedprocedure. Tokura, S.; Ueno, K.; Miyazaki, S.; Nishi, N., Molecularweight dependent antimicrobial activity by chitosan. Macromol. Symp.1997, 120, 1-9. Briefly, chitosan oligosaccharides (50 mg) weredissolved in water (2 mL) at pH 9.0. Rhodamine B isothiocyanate (RITC)was added to the solution in a 1:100 molar ratio to the primary amine ofthe chitosan oligosaccharides prior to the grafting of 2-methylaziridine. The solution was stirred at room temperature for 3 d in thedark. Subsequent dialysis and lyophilization yielded the RITC-labeledchitosan oligosaccharides.

By tuning the ratio of MAz and primary amine (e.g., 1:1 CSO 1 andChitosan 1, 2:1 CSO 2 and Chitosan 2), the number of MAz repeating unitsgrafted onto the chitosan oligosaccharides was tunable (supporting NMRdata), leading to a range of secondary amine concentrations and NOstorage. Acrylate-functionalized PEG chains were conjugated to theprimary amines on CSO 2 and Chitosan 2 by the Michael addition reactionto yield PEG-modified scaffolds (e.g., CSO 3 and Chitosan 3, See,schemes B and B′). Grafting of 2-methyl aziridine to theoligosaccharides increased the corresponding nitrogen content from 6.3to 8.9 and 10.8 wt % for CSO 1 and Chitosan 1 and for CSO 2 and Chitosan2, respectively. The PEGylation of CSO 2 and Chitosan 2 led to acorresponding decrease in nitrogen content (3.1 wt %) (CSO 3 andChitosan 3).

2. Nitric Oxide Charging, Storage and Release in FunctionalizedPolyglucosamines

The subject matter disclosed herein describes the optimization ofcharging condition for secondary-amine-functionalized chitosanoligosaccharides (e.g., CSO 1, 2, 3). Mixtures of methanol and waterwere used as the charging solvents. FIG. 4 shows the NO release profilesfor CSO 2-NO charged in different solvents. When the 7:3 methanol/waterwas used, the maximum total NO storage (e.g., ˜0.87 μmol/mg) wasyielded.

TABLE 3 Nitric oxide release characteristics in for secondaryamine-functionalized chitosan oligosaccharides (CSO 2; Chitosan 2/NO-5k)PBS (pH = 7.4) at 37° C. MeOH/H₂O 10:0 9:1 8:2 7:3 6:4 t[NO] (μmol/mg)0.58 ± 0.09 0.74 ± 0.12 0.81 ± 0.14 0.87 ± 0.16 0.75 ± 0.18 [NO]_(max)(ppb/mg) 2648 ± 120  4150 ± 70  4350 ± 484  5500 ± 414  5000 ± 572 Half-life (h) 2.40 ± 0.13 2.25 ± 0.02 2.05 ± 0.07 2.20 ± 0.14 2.05 ±0.25

The subject matter disclosed herein describes the control of total NOstorage by tuning the ratio of aziridine compounds to the primary amineson the chitosan oligosaccharides. By increasing the use of aziridinecompounds, greater secondary amine content and thus total NO storage canbe achieved. For example, as shown in FIGS. 5A and B and Tables 4A andB, the synthesis with 2-methyl aziridine/primary amines 2:1 ratioyielded total storage around 0.87 μmol/mg while the ratio 1:1 led to theNO storage of 0.30 μumol/mg. Accordingly, further increase of aziridinecompound usage would likely result in more enhanced NO storage. Themaximum NO storage (using the 7:3 methanol/water charging solvent ratio)was 0.87 μmol/mg, roughly 4× larger than that for previously reportedchitosan polysaccharides (˜0.2 μmol/mg). Du, J.; Hsieh, Y. L.Nanofibrous membranes from aqueous electrospinning of carboxymethylchitosan. Nanotechnology 2008, 19, 125707; Kim, S. K.; Rajapakse, N.Enzymatic production and biological activities of chitosanoligosaccharides (COS): A review. Carbohydr. Polym. 2005, 62, 357-368;Maghami (1988).

The subject matter disclosed herein describes the control of NO-releasekinetics by functionalizing the amine moieties of the secondaryamine-functionalized chitosan oligosaccharides (CSO 1,2). As shown inFIGS. 5A and B and Tables 4A and 4B, chitosan oligosaccharides modifiedwith hydrophilic PEG (CSO 3-NO) exhibited a greater initial NO flux andshorter half-life compared to the counterparts before PEGfunctionalization (CSO 2-NO).

TABLE 4A Nitric oxide-release properties of N-diazeniumdiolate NO donor-functionalized chitosan oligosaccharides. t[NO] [NO]_(max) t_(1/2)(μmol/mg) (ppb/mg) (h) CSO 1-NO 0.30 ± 0.04 1600 ± 215 3.60 ± 0.13 CSO2-NO 0.87 ± 0.16 5500 ± 414 2.20 ± 0.14 CSO 3-NO 0.35 ± 0.02 12600 ±2121 0.15 ± 0.01Accordingly, the oligosaccharide units will be present in mole ratiosthat are reflected in the NO release properties. In embodiments, m isfrom about 0.4 to about 0.9, for example about 0.4, 0.5, 0.6, 0.7, 0.8or 0.9; n is from about 0.1 to about 0.6, for example about 0.1, 0.2,0.3, 0.4, 0.5, or 0.6; wherein m and n represent the mole fraction ofeach unit and the sum of m and n is 1.

TABLE 4B Nitric oxide-release properties of different N-diazeniumdiolateNO donor-functionalized chitosan oligosaccharides in PBS (pH = 7.4, 37°C.) as measured using a chemiluminescence NO analyzer. t[NO]^(a)t[NO]^(b) [NO]_(max) t_(1/2) Material (μmol/mg) (μmol/mg) (ppb/mg) (h)Chitosan 1/ 0.30 ± 0.04 0.16 ± 0.03 1600 ± 215 3.60 ± 0.13 NO-5kChitosan 2/ 0.87 ± 0.16 0.52 ± 0.15 5500 ± 414 2.20 ± 0.14 NO-5kChitosan 3/ 0.35 ± 0.02 0.29 ± 0.01 12600 ± 2121 0.15 ± 0.01 NO-5kChitosan 2/ 0.84 ± 0.04 0.49 ± 0.02 7500 ± 550 2.06 ± 0.10 NO-2.5kChitosan 2/ 0.81 ± 0.05 0.47 ± 0.03 7350 ± 672 2.04 ± 0.05 NO-10k^(a)total NO released and ^(b)NO released over 24 and 4 h (μmol) permilligram of secondary amine-functionalized PPI. Each parameter wasanalyzed with multiple replicates (n = 3).N-diazeniumdiolate-functionalized chitosan oligosaccharides (1 mg) (CSO1-NO, Chitosan 1/NO, CSO 2-NO, Chitosan 2/NO, CSO 3-NO, Chitosan 3/NO)in the water/methanol mixture were added into a sample vessel containing30 mL deoxygenated phosphate buffered saline (PBS) (10 mM, pH=7.4) at37° C., which initiated NO release. To quantify the NO released, thesolution was purged with nitrogen at a flow rate of 70 mL/min to carrythe liberated NO to the analyzer. Additional nitrogen flow was suppliedto the vessel to match the collection rate of the instrument (200mL/min). The analysis of NO was terminated when the NO release levelsfell to below 10 ppb NO/mg chitosan oligosaccharides. Chemiluminescencedata for the NO-releasing chitosan oligosaccharides were representedas: 1) total amount of NO release (t[NO], μmol NO/mg of secondaryamine-functionalized chitosan oligosaccharides); 2) the maximum flux ofNO release ([NO]max, ppb/mg of secondary amine-functionalized chitosanoligosaccharides); and 3) the half-life of NO release (t_(1/2)).

3. Mouse Fibroblast Viability Assay

L929 mouse fibroblasts were grown in DMEM supplemented with 10% (v/v)fetal bovine serum (FBS) and 1 wt % penicillin/streptomycin, andincubated in 5% (v/v) CO₂ under humidified conditions at 37° C. Afterreaching 80% confluency, the cells were trypsinized, seeded ontotissue-culture treated polystyrene 96-well plates at a density of 3×10⁴cells/mL and incubated at 37° C. for 48 h. The supernatant was thenaspirated prior to adding 200 μL fresh DMEM and 50 μL of a NO-releasingchitosan oligosaccharides solution in PBS to each well. After incubationat 37° C. for 24 h, the supernatant was aspirated and 120 μL mixture ofDMEM/MTS/PMS (105/20/1, v/v/v) was added to each well. The absorbance ofthe resulting colored solution after 1.5 h incubation at 37° C. wasquantified at 490 nm using a Thermoscientific Multiskan EX plate reader.The mixture of DMEM/MTS/PMS and untreated cells were used as blank andcontrol, respectively. The cell viability was calculated by equation 1.

$\begin{matrix}{{{Cell}\mspace{14mu}{Viability}} = \frac{( {{Absorbance}_{{treated}\mspace{14mu}{cell}} - {Absorbance}_{blank}} )}{( {{Absorbance}_{{untreated}\mspace{14mu}{cell}} - {Absorbance}_{blank}} )}} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

4. The cytotoxicity of control and NO-releasing chitosanoligosaccharides were compared by exposing mouse fibroblast cells to theoligosaccharides at the MBCs against P. aerugionsa biofilms noted above.The results of the normalized cell viabilities of control andNO-releasing chitosan oligosaccharides after 24 h incubation are shownin FIGS. 8A and B. Regardless of size (i.e., molecular weight), thecontrol and NO-releasing chitosan oligosaccharides were non-toxicagainst mouse fibroblast cells at the MBCs for the NO-releasingscaffolds, indicating an advantage of these materials as anti-biofilmagents compared to other antibacterial agents. The NO-releasing chitosanoligosaccharides exhibited lower cytotoxicity than the chitosancontrols.

5. Bactericidal Assays Under Static Conditions

P. aeruginosa bacterial cultures were grown from a frozen (−80° C.)stock overnight in TSB at 37° C. A 500 μL aliquot of the resultingsuspension was added into 50 mL fresh TSB and incubated at 37° C. for ˜2h until the concentration reached ˜1×10⁸ colony forming units (CFU)/mL,as confirmed by the OD600, replicate plating and enumeration on nutrientagar. A working bacterial stock was generated by plating the bacterialsuspension on TSA and incubating at 37° C. overnight. The TSA bacterialstocks were prepared weekly and stored at 4° C. For bactericidal assays,colonies of P. aeruginosa were taken from the TSA plate, dispersed in 3mL TSB, and incubated at 37° C. overnight. A 500 μL aliquot of culturewas added to 50 mL fresh TSB and incubated to a concentration of ˜1×10⁸CFU/mL. The bacteria was collected by centrifugation, resuspended inPBS, and diluted to ˜1×10⁶ CFU/mL. The bactericidal efficacy ofNO-releasing chitosan oligosaccharides against P. aeruginosa wasevaluated by incubating the bacteria suspension with NO-releasingchitosan oligosaccharides at 37° C. At 4 h, 100 μL, aliquots of thebacterial suspensions were removed, diluted 10-fold in PBS, plated onTSA, and incubated overnight at 37° C. The minimum concentration ofNO-releasing chitosan oligosaccharides that resulted in a 3-logreduction of bacterial viability was defined as the minimum bactericidalconcentration (MBC) for planktonic studies.

Bacterial viability assays were performed under static conditions todetermine the concentration of chitosan required to reduce bacteriaviability by 3 logs (i.e., 99.9% killing), which hereafter will bereferred to as the minimum bactericidal concentration or MBC. The amountof NO delivered from NO-releasing chitosan oligosaccharides (Table 4)over the time of the assay (4 h) was also examined to quantitativelyassess the NO dose necessary for 99.9% bacterial killing. Both MBCs andthe bactericidal NO doses required for the chitosan oligosaccharides areprovided in Table 5.

TABLE 5 Minimum bactericidal concentration (MBC) and NO doses ofNO-releasing chitosan oligosaccharides for 3-log reduction in planktonicP. aeruginosa viability. Chitosans MBC (μg/mL) NO dose (μmol/mL)Chitosan 1/NO-5k 2000 0.32 Chitosan 2/NO-5k 200 0.10 Chitosan 3/NO-5k1500 0.45 Chitosan 2/NO-2.5k 250 0.12Regardless of size (i.e., molecular weight of about 2.5, 5, or 10 kDa),each of the NO-releasing chitosan oligosaccharides (Chitosan 2/NO-2.5 k,Chitosan 2/NO-5 k, Chitosan 2/NO-10 k) exhibited similar bactericidal NOconcentrations (i.e., ˜10 μmol NO/mL) for 3-log killing (Table 5). Eachof the NO-releasing chitosan oligosaccharides studied (including CSO2-NO-5k) resulted in ≥99.9% killing of P. aeruginosa. At equivalentconcentrations, the control (non-NO-releasing) chitosan did not lead toa significant reduction in bacterial viability, indicating NO as thebactericidal agent (data not shown).

6. Treatment of P. aeruginosa Biofilms with NO-releasing ChitosanOligosaccharides

A CDC bioreactor (Biosurface Technologies, Bozeman, Mont.) was used togrow P. aeruginosa biofilms over a 48 h period. Briefly, medical gradesilicone rubber substrates were mounted in coupon holders prior toassembling the reactor. The assembled reactor was then autoclaved. Thereactor effluent line was clamped, and 1% (v/v) sterile TSB (500 mL) wasadded aseptically. P. aeruginosa was then cultured in TSB to 108 CFU/mL.The reactor was inoculated with an aliquot (1 mL) of this bacterialsuspension at a final concentration ˜2×105 CFU/mL. The reactor wasincubated at 37° C. for 24 h with slow stirring (150 rpm). Followingthis “batch phase” growth, the reactor media was refreshed continuouslywith 0.33% (v/v) TSB at 6 mL/min for another 24 h through the effluentline.

P. aeruginosa biofilms grown on silicone rubber substrates were exposedto chitosan oligosaccharide in PBS with slight agitation (37° C., 24 h)to determine the minimum bactericidal concentration (MBC) necessary toelicit a 5-log reduction in viability. At 24 h, samples were thensonicated and vortexed to disrupt the biofilm. Aliquots (100 μL) of thebacteria/chitosan suspensions were diluted and plated on TSA. Afterincubating the TSA plates overnight at 37° C., bacteria viability wasdetermined by counting observed colonies. Of note, the limit ofdetection for this selected plate counting method is 2.5×103 CFU/mL. Assuch, biofilm growth conditions were selected to accurately represent a5-log reduction in viability for biofilms.

To evaluate the anti-biofilm activity of NO-releasing chitosanoligosaccharides (e.g., Chitosan 1/NO-5k, Chitosan 2/NO-5k, Chitosan3/NO-5k), P. aeruginosa biofilms were exposed to 0.2-1.3 mg/mLNO-releasing chitosan oligosaccharides for 24 h (corresponding to˜0.17-0.46 μmol NO/mL). After treatment, the biofilms were removed fromthe silicone rubber substrates by vortexing and sonication to enableviability quantification. Salmon, D. J.; Torres de Holding, C. L.;Thomas, L.; Peterson, K. V.; Goodman, G. P.; Saavedra, J. E.;Srinivasan, A.; Davies, K. M.; Keefer, L. K.; Miranda, K. M. HNO and NOrelease from a primary amine-based diazeniumdiolate as a function of pH.Inorg. Chem. 50, 3262-70. Control experiments were performed to confirmthe growth of P. aeruginosa biofilms using the selected protocol. Asshown in FIG. 7, the viability of P. aeruginosa in the biofilm was˜2×10⁸ CFU when exposed only to PBS. The chitosan concentrations for5-log reduction of biofilm bacteria viability (MBC) were 400, 700, and1000 μg/mL for Chitosan 2/NO-5k, Chitosan 1/NO-5k, and Chitosan 3/NO-5k,respectively. Chitosan 2/NO-5k exhibited the greatest anti-biofilmefficacy, a likely result due to both increased NO storage/release andrapid association with the negatively charged bacteria. AlthoughChitosan 1/NO-5k and Chitosan 3/NO-5 k stored similar levels of NO (˜0.3μmol/mg), Chitosan 1/NO-5k was more effective at eradicating the biofilmbacteria (MBC 700 μg/mL) compared to Chitosan 3/NO-5k (MBC 1000 μg/mL).The association of Chitosan 2/NO-5k and Chitosan 3/NO-5k with P.aeruginosa biofilm was evaluated using confocal microscope. As shown inFIG. 9A-F, biofilms exposed to Chitosan 2/NO-5k exhibited more intensered fluorescence compared to Chitosan 3/NO-5k, again confirming theenhanced association of the positively charged Chitosan 2/NO-5k with thebacteria.

7. Confocal Microscopy

P. aeruginosa was cultured in TSB to a concentration of ˜1×10^(x)CFU/mL, collected via centrifugation (3645×g for 10 min), resuspended insterile PBS, and adjusted to ˜1×10⁶ CFU/mL. Aliquots of the bacteriasolution were incubated in a glass bottom confocal dish for 1.5 h at 37°C. A Zeiss 510 Meta inverted laser scanning confocal microscope with a543 nm HeNe excitation laser and a LP 585 nm filter was used to obtainfluorescence images of the rhodamine B isothiocyanate (RITC)-modifiedchitosan oligosaccharides. The bright field and fluorescence images werecollected by a N.A. 1.2 C-apochromat water immersion lens with a 40×objective. Solutions of RITC-labeled NO-releasing chitosanoligosaccharides in PBS (1.5 mL) were added to the bacteria solution(1.5 mL) in the glass confocal dish to achieve a final concentration of150 μg/mL. Images were collected every 2 min to characterize theassociation, if any, of the chitosan oligosaccharides with P. aeruginosatemporally. To observe the association of chitosan oligosaccharides withbacteria within biofilms, Established biofilms stained with syto 9 (10μM) were incubated with RITC-labeled chitosan oligosaccharides (150μg/mL in PBS) for 2.5 h. Prior to imaging, samples were rinsed with PBS(3×). A Zeiss 510 Meta inverted laser scanning confocal microscope with488 nm Ar and 543 nm HeNe excitation lasers, and a BP 505-530 nm and LP585 nm filters, respectively, was used to obtain all confocal images.Fluorescence images were collected with a 20× objective.

Confocal microscopy was utilized to compare the association kinetics ofChitosan 3/NO-5k and Chitosan 2/NO-5k with bacteria. Rhodamine Bisothiocyanate (RITC)-labeled Chitosan 2/NO-5k and Chitosan 3/NO-5k weresynthesized. Maghami (1988). The potential impact of RITC onchitosan-bacteria association was minimized by using small concentrationof RITC (i.e., in 1:100 molar ratio to total primary amines). The degreeof association of the NO-releasing chitosan oligosaccharides withbacteria was then followed by measuring red fluorescence surrounding thebacteria. Chitosan 2/NO-5k associated with the bacteria more rapidly(within 24 min) than Chitosan 3/NO-5k (86 min) (FIG. 10). Thefluorescence from Chitosan 2/NO-5k at 42 min was significantly greaterthan that of Chitosan 3/NO-5k at 110 min, further demonstrating thatChitosan 3/NO-5k associated with the bacteria at a much slower rate dueto the PEG (neutral) modification. Further inspection of Chitosan2/NO-5k and Chitosan 3/NO-5k association with P. aeruginosa revealedenhanced bacteria association for Chitosan 2/NO-5k (FIG. 10-G, H).

Although chitosan molecular weight was not observed to play asignificant role in planktonic killing, less effective bacteria killingwas observed when using Chitosan 2/NO-10k, the largest chitosanoligosaccharides (600 μg/mL vs. 400 μg/mL for Chitosan 2/NO-10k andChitosan 2/NO-2.5k) against biofilms. The efficient association ofchitosan oligosaccharides with bacteria in biofilms is advantageous inview of previously reported NO-releasing polysaccharides which areinsoluble under physiological conditions. All documents cited orreferenced in the application cited documents, and all documents citedor referenced herein (“herein cited documents”), and all documents citedor referenced in herein cited documents, together with anymanufacturer's instructions, descriptions, product specifications, andproduct sheets for any products mentioned herein or in any documentincorporated by reference herein, are hereby incorporated herein byreference, and may be employed in the practice of the invention.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

That which is claimed is:
 1. A polyglucosamine comprising, at least onestructural unit:

and optionally, at least one structural unit:

wherein, i. R₁, R₂, R₃, R₄, R₅, and R₆, are each hydrogen; ii.

is a single bond; iii. Q is —(CR_(c)R_(d))_(v)—; wherein R_(c) and R_(d)are hydrogen and v is 2; iv. p is an integer from 1 to 10; v. A is

wherein, L is N and G is hydrogen; vi. X is taken together with N toform a nitric oxide donor; vii. B is hydrogen; and viii. D is—NR_(a)R_(b), wherein R_(a) and R_(b) are hydrogen.
 2. Thepolyglucosamine of claim 1, wherein p is
 1. 3. The polyglucosamine ofclaim 1, wherein the nitric oxide donor taken together with the atom onthe polyglucosamine to which it is bound is selected from the groupconsisting of a diazeniumdiolate, nitrosothiol, a nitrosamine, ahydroxyl nitrosamine, a hydroxyl amine, a hydroxyurea, and combinationthereof.
 4. The polyglucosamine of claim 1, wherein the nitric oxidedonor is diazeniumdiolate.
 5. The polyglucosamine of claim 1, whereinthe polyglucosamine is water soluble.
 6. The polyglucosamine of claim 1,wherein the polyglucosamine is water soluble such that >50 mg of thepolyglucosamine dissolves per mL of water.
 7. The polyglucosamine ofclaim 1, wherein the polyglucosamine is water soluble such that >75 mgof the polyglucosamine dissolves per mL of water.
 8. The polyglucosamineof claim 1, wherein the polyglucosamine is water soluble such that >100mg of the polyglucosamine dissolves per mL of water.
 9. Thepolyglucosamine of claim 1, wherein the viscosity average molecularweight (M_(v)) as determined by the equation [η]=1.81×10⁻³ M_(v) ^(0.93)is between about 2000 and about 11000 g/mol.
 10. The polyglucosamine ofclaim 1, wherein the viscosity average molecular weight (My) asdetermined by the equation [η]=1.81×10⁻³ M_(v) ^(0.93) is between about3000 and about 6000 g/mol.
 11. The polyglucosamine of claim 1, whereinthe elemental composition is: between about 40% and about 50% C; betweenabout 6% and about 9% H; and between about 3% and about 11% N.
 12. Thepolyglucosamine of claim 1, wherein the elemental composition is:between about 42% and about 51% C; between about 6% and about 9% H; andbetween about 3% and about 11% N.
 13. The polyglucosamine of claim 1,wherein the polyglucosamine includes greater than about 0.3 μmol/mg ofreleasable nitric oxide.
 14. The polyglucosamine of claim 1, wherein thepolyglucosamine includes greater than about 0.8 μmol/mg of releasablenitric oxide.
 15. A method of delivering nitric oxide to a subject,comprising: administering an effective amount of the polyglucosamine ofclaim 1 to the subject.
 16. A method of treating a disease state,comprising: administering an effective amount of the polyglucosamine ofclaim 1 to a subject in need thereof, wherein the disease state isselected from the group consisting of a cancer, a cardiovasculardisease, a microbial infection; platelet aggregation and plateletadhesion caused by the exposure of blood to a medical device;pathological conditions resulting from abnormal cell proliferation;transplantation rejections, autoimmune diseases, inflammation, vasculardiseases; scar tissue; wound contraction, restenosis, pain, fever,gastrointestinal disorders, respiratory disorders, sexual dysfunctions,and sexually transmitted diseases.
 17. The method of claim 16, whereinsaid disease state is cystic fibrosis.
 18. A pharmaceutical formulationcomprising: the polyglucosamine of claim 1; and a pharmaceuticallyacceptable carrier.